Duplexes Formed by G₄C₂ Repeats Contain Alternate Slow- and Fast-Flipping G·G Base Pairs

Abstract

Aberrant expansion of the hexanucleotide GGGGCC (or G4C2) repeat in the human C9ORF72 gene is the most common genetic factor found behind amyotrophic lateral sclerosis and frontotemporal dementia. The hypothesized pathways, through which the repeat expansions contribute to the pathology, involve one or more secondary structural forms of the DNA and/or RNA sequences, such as G-quadruplexes, duplexes, and hairpins. Here, we study the structures of DNA and RNA duplexes formed by G4C2 repeats, which contain G­(syn)·G­(anti) base pairs flanked by either G·C or C·G base pairs. We show that duplexes formed by G4C2 repeats contain alternately two types of G·G pair contexts exhibiting different syn–anti base flipping dynamics (∼100 ms vs ∼2 ms for DNA and ∼50 ms vs ∼20 ms for RNA at 10 °C, respectively) depending on the flanking bases, with the slow-flipping G·G pairs being flanked by a guanine at the 5′-end and the fast-flipping G·G pairs being flanked by a cytosine at the 5′-end. Our findings on the structures and dynamics of G·G base pairs in DNA and RNA duplexes formed by G4C2 repeats provide a foundation for further studies of the functions and targeting of such biologically relevant motifs.

Role of C9orf72 hexanucleotide repeat expansions in ALS/FTD pathogenesis

“…It has been proved that the transcribed RNA from G4C2 repeat expansions [termed r(G4C2)n] can indeed form various structures, including G-quadruplexes ( Fratta et al, 2012 ; Reddy et al, 2013 ; Haeusler et al, 2014 ), hairpin structures ( Su et al, 2014 ) and duplex structures ( Maity et al, 2021 ). These diverse structures have been confirmed through topological investigations using gel-shift assay, CD spectroscopy and NMR spectroscopy.…”

Advances in the Structure of GGGGCC Repeat RNA Sequence and Its Interaction with Small Molecules and Protein Partners

“…Nuclear Magnetic Resonance (NMR) spectra are able to provide more structural details, some of which yield full structural determination or provide evidence of co-existence of different conformations (Figure 2a). Depending on the sequence length and solution conditions, r(GGGGCC) n can adopt different secondary structures, including G4 [58,59] and hairpin conformations [60]. In 2012, Adrian M. Isaacs and colleagues demonstrated that r(GGGGCC) 3 GGGGC can fold into G4 or double-stranded structures, with the topology being influenced by the presence of cation ions in the solution [61].…”

Conformational Dynamics of RNA G4C2 and C2G4 Repeat Expansions causing ALS/FTD using NMR and Molecular Dynamics Studies

“…r(G4C2) exp adopts multiple structures, including Gquadruplexes 27 and hairpin structures 13 , the latter of which contains an array of 1×1 G/G internal loop conformations (Figure 1B). It has been shown that 1×1 G/G internal loops are very dynamic and can interconvert between syn-anti and anti-syn (syn-anti ↔ anti-syn) 28 .…”

“…Depending on the complexity and dynamics of the target, different experimental techniques such as X-ray crystallography, NMR spectroscopy, and cryogenic electron microscopy (cryo-EM) can be used to discover the atomistic details of a structure. For example, 1×1 G/G internal loops have been recently studied by NMR spectroscopy, where it was shown that syn ↔ anti base flipping time in r(G4C2) exp depends on the neighboring GC base pairs due to stacking interactions displaying the importance of closing base pairs in RNA loop dynamics 28. These structural rearrangements are important in target recognition as shown in binding mode of DNA mismatch repair enzyme MutS with 1×1 G/G mismatches73 and in discovery of biomarkers and lead compounds targeting r(G4C2) exp74 .…”